Since the development of the Edmonton protocol, islet transplantation is increasingly encouraging as a treatment for type 1 diabetes. Strategies to ameliorate problems with the intraportal site include macroencapsulating the islets in diverse biomaterials. Characterization of these biomaterials is important to optimally tune the properties to support islets and promote vascularization. In this study, we characterize the cross-linker-dependent properties of collagen-based matrices containing chondroitin-6-sulfate, chitosan, and laminin, cross-linked with 7.5, 30, or 120 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The swelling ratio was found to be significantly negatively correlated with increasing cross-linker concentrations (p<0.0001; R2=0.718). The matrix released insulin in a reproducible logarithmic manner (R2 of 0.99 for all concentrations), demonstrating cross-linker-dependent control of drug release. The matrices with the highest cross-linker concentrations resisted degradation by collagenase for longer than the lowest concentrations (58.13%±2.22% vs. 13.69%±7.67%; p<0.05). Scanning electron microscopy images of the matrices revealed that the matrices had uniform topography and porosity, indicating efficient cross-linking and incorporation of the polymer components. Matrices were transplanted subcutaneously in naive BALB/c mice, and the number and size of vessels were quantified using von Willebrand factor staining; matrices with higher cross-linking concentrations had significantly larger capillaries at every time point up to 4 weeks after transplantation compared to the lowest cross-linker concentration group. CD31 staining visualized the capillaries at each time point. Taken together, these data show that this collagen-based matrix is reproducible with cross-linking-dependent properties that can be optimized to support vascularization and islet function.

Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD).

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in preclinical models. In the type 1 diabetes (T1D) research field, multiple lines of nonobese diabetic (NOD) mice have been engineered to express Cre recombinase in pancreatic β cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background have high β-cell specificity with no reported off-target effects. We explored whether NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in T1D disease modeling. We studied wild-type (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knockin in Ins1Neo/Cre mice were not additive to the Cre knockin alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at age 12 weeks. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has important implications for the experimental design and interpretation of preclinical T1D studies using β-cell-selective Cre in NOD mice.

miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, β-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of β-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-β signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how β-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.

Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre+LeprloxTB/loxTB) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre-LeprloxTB/loxTB). In contrast, Lyz2Cre+LeprloxTB/loxTB females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre+LeprloxTB/loxTB males had a mildly improved plasma glucose profile compared to Cre- controls while Lyz2Cre+LeprloxTB/loxTB females had a similar glucose excursion to their Cre- controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre+Leprflox/flox) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre+LeprloxTB/loxTB mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre+Leprflox/flox mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.

The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells.

The relative contribution of peripheral and central leptin signalling to the regulation of metabolism and the mechanisms through which leptin affects glucose homeostasis have not been fully elucidated. We generated complementary lines of mice with either leptin receptor (Lepr) knockdown or reconstitution in adipose tissues using Cre-lox methodology. Lepr knockdown mice were modestly lighter and had lower plasma insulin concentrations following an oral glucose challenge compared to controls, despite similar insulin sensitivity. We rendered male mice diabetic using streptozotocin (STZ) and found that upon prolonged leptin therapy, Lepr knockdown mice had an accelerated decrease in blood glucose compared to controls that was associated with higher plasma concentrations of leptin and leptin receptor. Mice with transcriptional blockade of Lepr (LeprloxTB/loxTB) were obese and hyperglycemic and reconstitution of Lepr in adipose tissues of LeprloxTB/loxTB mice resulted in males reaching a higher maximal body weight. Although mice with adipose tissue Lepr reconstitution had lower blood glucose levels at several ages, their plasma insulin concentrations during an oral glucose test were elevated. Thus, attenuation or restoration of Lepr in adipocytes alters the plasma insulin profile following glucose ingestion, modifies the glucose-lowering effect of prolonged leptin therapy in insulin-deficient diabetes, and may modulate weight gain.

Excess circulating insulin is associated with obesity in humans and in animal models. However, the physiologic causality of hyperinsulinemia in adult obesity has rightfully been questioned because of the absence of clear evidence that weight loss can be induced by acutely reversing diet-induced hyperinsulinemia. Herein, we describe the consequences of inducible, partial insulin gene deletion in a mouse model in which animals have already been made obese by consuming a high-fat diet. A modest reduction in insulin production/secretion was sufficient to cause significant weight loss within 5 wk, with a specific effect on visceral adipose tissue. This result was associated with a reduction in the protein abundance of the lipodystrophy gene polymerase I and transcript release factor ( Ptrf; Cavin) in gonadal adipose tissue. RNAseq analysis showed that reduced insulin and weight loss also associated with a signature of reduced innate immunity. This study demonstrates that changes in circulating insulin that are too fine to adversely affect glucose homeostasis nonetheless exert control over adiposity.-Page, M. M., Skovsø, S., Cen, H., Chiu, A. P., Dionne, D. A., Hutchinson, D. F., Lim, G. E., Szabat, M., Flibotte, S., Sinha, S., Nislow, C., Rodrigues, B., Johnson, J. D. Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain.

Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas. However, it is not clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in β-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of β-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single β-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH β-cells were enriched for markers of β-cell maturity. Ins2(GFP)HIGH β-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity.

Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined.

Antiadiposity effects of caloric restriction (CR) are associated with reduced insulin/IGF-1 signaling, but it is unclear whether the effects of CR would be additive to genetically reducing circulating insulin. To address this question, we examined female Ins1(+/-):Ins2(-/-) mice and Ins1(+/+):Ins2(-/-) littermate controls on either an ad libitum or 60% CR diet. Although Igf1 levels declined as expected, CR was unable to reduce plasma insulin levels in either genotype below their ad libitum-fed littermate controls. In fact, 53-week-old Ins1(+/-):Ins2(-/-) mice exhibited a paradoxical increase in circulating insulin in the CR group compared with the ad libitum-fed Ins1(+/-):Ins2(-/-) mice. Regardless of insulin gene dosage, CR mice had lower fasting glucose and improved glucose tolerance. Although body mass and lean mass predictably fell after CR initiation, we observed a significant and unexpected increase in fat mass in the CR Ins1(+/-):Ins2(-/-) mice. Specifically, inguinal fat was significantly increased by CR at 66 weeks and 106 weeks. By 106 weeks, brown adipose tissue mass was also significantly increased by CR in both Ins1(+/-):Ins2(-/-) and Ins1(+/+):Ins2(-/-) mice. Interestingly, we observed a clear whitening of brown adipose tissue in the CR groups. Mice in the CR group had altered daily energy expenditure and respiratory exchange ratio circadian rhythms in both genotypes. Multiplexed analysis of circulating hormones revealed that CR was associated with increased fasting and fed levels of the obesogenic hormone, glucose-dependent insulinotropic polypeptide. Collectively these data demonstrate CR has paradoxical effects on adipose tissue growth in the context of genetically reduced insulin.

Pancreatic β cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in β cells, but few studies have examined male-female differences in β cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in β cell and islet gene expression and function using both human and rodent samples.

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsrKO and βInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female βInsrKO and βInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.